The compounds of the present invention are antagonists of the VLA-4 integrin (xe2x80x9cvery late antigen-4xe2x80x9d; CD49d/CD29; or xcex14xcex21), the xcex14xcex27 integrin (LPAM-1 and xcex14xcex2p), and/or the xcex19xcex21 integrin, thereby blocking the binding of VLA-4 to its various ligands, such as VCAM-1 and regions of fibronectin, xcex14xcex27 to its various ligands, such as MadCAM-1, VCAM-1 and fibronectin, and /or xcex19xcex21 to its various ligands, such as tenascin, osteopontin and VCAM-1. Thus, these antagonists are useful in inhibiting cell adhesion processes including cell activation, migration, proliferation and differentiation. These antagonists are useful in the treatment, prevention and suppression of diseases mediated by VLA-4, xcex14xcex27-, and/or xcex19xcex21-binding and cell adhesion and activation, such as AIDS-related dementia, allergic conjunctivitis, allergic rhinitis, Alzheimer""s disease, aortic stenosis, asthma, atherosclerosis, autologous bone marrow transplantation, certain types of toxic and immune-based nephritis, contact dermal hypersensitivity, inflammatory bowel disease including ulcerative colitis and Crohn""s disease, inflammatory lung diseases, inflammatory sequelae of viral infections, meningitis, multiple sclerosis, myocarditis, organ transplantation, psoriasis, restenosis, retinitis, rheumatoid arthritis, septic arthritis, stroke, tumor metastasis, type I diabetes, vascular occlusion following angioplasty.
The present invention relates to substituted urea derivatives which are useful for the inhibition and prevention of leukocyte adhesion and leukocyte adhesion-mediated pathologies. This invention also relates to compositions containing such compounds and methods of treatment using such compounds.
Many physiological processes require that cells come into close contact with other cells and/or extracellular matrix. Such adhesion events may be required for cell activation, migration, proliferation and differentiation. Cell-cell and cell-matrix interactions are mediated through several families of cell adhesion molecules (CAMs) including the selectins, integrins, cadherins and immunoglobulins. CAMs play an essential role in both normal and pathophysiological processes. Therefore, the targetting of specific and relevant CAMs in certain disease conditions without interfering with normal cellular functions is essential for an effective and safe therapeutic agent that inhibits cell-cell and cell-matrix interactions.
The integrin superfamily is made up of structurally and functionally related glycoproteins consisting of a and b heterodimeric, transmembrane receptor molecules found in various combinations on nearly every mammalian cell type. (for reviews see: E. C. Butcher, Cell, 67, 1033 (1991); T. A. Springer, Cell, 76, 301 (1994); D. Cox et al., xe2x80x9cThe Pharmacology of the Integrins.xe2x80x9d Medicinal Research Rev. 14, 195 (1994) and V. W. Engleman et al., xe2x80x9cCell Adhesion Integrins as Pharmaceutical Targets.xe2x80x9d in Ann. Repts. in Medicinal Chemistry, Vol. 31, J. A. Bristol, Ed.; Acad. Press, NY, 1996, p. 191).
VLA-4 (xe2x80x9cvery late antigen-4xe2x80x9d; CD49d/CD29; or xcex14xcex21) is an integrin expressed on all leukocytes, except platelets and mature neutrophils, including dendritic cells and macrophage-like cells and is a key mediator of the cell-cell and cell-matrix interactions of of these cell types (see M. E. Hemler, xe2x80x9cVLA Proteins in the Integrin Family: Structures, Functions, and Their Role on Leukocytes.xe2x80x9d Ann. Rev. Immunol. 8, 365 (1990)). The ligands for VLA-4 include vascular cell adhesion molecule-1 (VCAM-1) and the CS-1 domain of fibronectin (FN). VCAM-1 is a member of the Ig superfamily and is expressed in vivo on endothelial cells at sites of inflammation. (See R. Lobb et al. xe2x80x9cVascular Cell Adhesion Molecule 1.xe2x80x9d in Cellular and Molecular Mechanisms of Inflammation, C. G. Cochrane and M. A. Gimbrone, Eds.; Acad. Press, San Diego, 1993, p. 151.) VCAM-1 is produced by vascular endothelial cells in response to pro-inflammatory cytokines (See A. J. H. Gearing and W. Newman, xe2x80x9cCirculating adhesion molecules in disease.xe2x80x9d, Immunol. Today, 14, 506 (1993). The CS-1 domain is a 25 amino acid sequence that arises by alternative splicing within a region of fibronectin. (For a review, see R. O. Hynes xe2x80x9cFibronectins.xe2x80x9d, Springer-Velag, N.Y., 1990.) A role for VLA-4/CS-1 interactions in inflammatory conditions has been proposed (see M. J. Elices, xe2x80x9cThe integrin xcex14xcex21 (VLA-4) as a therapeutic targetxe2x80x9d in Cell Adhesion and Human Disease, Ciba Found. Symp., John Wiley and Sons, N.Y., 1995, p. 79).
xcex14xcex27 (also referred to as LPAM-1 and xcex14xcex2p) is an integrin expressed on leukocytes and is a key mediator of leukocyte trafficking and homing in the gastrointestinal tract (see C. M. Parker et al., Proc. Natl. Acad. Sci. USA, 89, 1924 (1992)). The ligands for xcex14xcex27 include mucosal addressing cell adhesion molecule-1 (MadCAM-1) and, upon activation of xcex14xcex27, VCAM-1 and fibronectin (Fn). MadCAM-1 is a member of the Ig superfamily and is expressed in vivo on endothelial cells of gut-associated mucosal tissues of the small and large intestine (xe2x80x9cPeyer""s Patchesxe2x80x9d) and lactating mammary glands. (See M. J. Briskin et al., Nature, 363, 461 (1993); A. Hamann et al., J. Immunol., 152, 3282 (1994)). MadCAM-1 can be induced in vitro by proinflammatory stimuli (See E. E. Sikorski et al. J. Immunol., 151, 5239 (1993)). MadCAM-1 is selectively expressed at sites of lymphocyte extravasation and specifically binds to the integrin, xcex14xcex27.
The xcex19xcex21 integrin is found on airway smooth muscle cells, non-intestinal epithelial cells (see Palmer et al., J. Cell Biol., 123, 1289 (1993)), and neutrophils, and, less so, on hepatocytes and basal keratinocytes (see Yokosaki et al., J. Biol. Chem., 269, 24144 (1994)). Neutrophils, in particular, are intimately involved in acute inflammatory repsonses. Attenuation of neutrophil involvement and/or activation would have the effect of lessening the inflammation. Thus, inhibition of xcex19xcex21 binding to its respective ligands would be expected to have a positive effect in the treatment of acute inflammatory conditions.
Neutralizing anti-xcex14 antibodies or blocking peptides that inhibit the interaction between VLA-4 and/or xcex14xcex27 and their ligands have proven efficacious both prophylactically and therapeutically in several animal models of disease, including i) experimental allergic encephalomyelitis, a model of neuronal demyelination resembling multiple sclerosis (for example, see T. Yednock et al., xe2x80x9cPrevention of experimental autoimmune encephalomyelitis by antibodies against xcex14xcex21 integrin.xe2x80x9d Nature, 356, 63 (1993) and E. Keszthelyi et al., xe2x80x9cEvidence for a prolonged role of xcex14 integrin throughout active experimental allergic encfephalomyelitis.xe2x80x9d Neurology, 47, 1053 (1996)); ii) bronchial hyperresponsiveness in sheep and guinea pigs as models for the various phases of asthma (for example, see W. M. Abraham et al., xe2x80x9cxcex14-Integrins mediate antigen-induced late bronchial responses and prolonged airway hyperresponsiveness in sheep.xe2x80x9d J. Clin. Invest. 93, 776 (1993) and A. A. Y. Milne and P. P. Piper, xe2x80x9cRole of VLA-4 integrin in leucocyte recruitment and bronchial hyperresponsiveness in the gunea-pig.xe2x80x9d Eur. J. Pharmacol., 282, 243 (1995)); iii) adjuvant-induced arthritis in rats as a model of inflammatory arthritis (see C. Barbadillo et al., xe2x80x9cAnti-VLA-4 mAb prevents adjuvant arthritis in Lewis rats.xe2x80x9d Arthr. Rheuma. (Suppl.), 36 95 (1993) and D. Seiffge, xe2x80x9cProtective effects of monoclonal antibody to VLA-4 on leukocyte adhesion and course of disease in adjuvant arthritis in rats.xe2x80x9d J. Rheumatol., 23, 12 (1996)); iv) adoptive autoimmune diabetes in the NOD mouse (see J. L. Baron et al., xe2x80x9cThe pathogenesis of adoptive murine autoimmune diabetes requires an interaction between xcex14-integrins and vascular cell adhesion molecule-1.xe2x80x9d, J. Clin. Invest., 93, 1700 (1994), A. Jakubowski et al., xe2x80x9cVascular cell adhesion molecule-Ig fusion protein selectively targets activated xcex14-integrin receptors in vivo: Inhibition of autoimmune diabetes in an adoptive transfer model in nonobese diabetic mice.xe2x80x9d J. inmunol., 155, 938 (1995), and X. D. Yang et al., xe2x80x9cInvolvement of beta 7 integrin and mucosal addressin cell adhesion molecule-1 (MadCAM-1) in the development of diabetes in nonobese diabetic micexe2x80x9d, Diabetes, 46, 1542 (1997)); v) cardiac allograft survival in mice as a model of organ transplantation (see M. Isobe et al., xe2x80x9cEffect of anti-VCAM-1 and anti-VLA-4 monoclonal antibodies on cardiac allograft survival and response to soluble antigens in mice.xe2x80x9d, Tranplant. Proc., 26, 867 (1994) and S. Molossi et al., xe2x80x9cBlockade of very late antigen-4 integrin binding to fibronectin with connecting segment-1 peptide reduces accelerated coronary arteripathy in rabbit cardiac allografts.xe2x80x9d J. Clin Invest., 95, 2601 (1995)); vi) spontaneous chronic colitis in cotton-top tamarins which resembles human ulcerative colitis, a form of inflammatory bowel disease (see D. K. Podolsky et al., xe2x80x9cAttenuation of colitis in the Cotton-top tamarin by anti-xcex14 integrin monoclonal antibody.xe2x80x9d, J. Clin. Invest., 92, 372 (1993)); vii) contact hypersensitivity models as a model for skin allergic reactions (see T. A. Ferguson and T. S. Kupper, xe2x80x9cAntigen-independent processes in antigen-specific immunity.xe2x80x9d, J. Immunol., 150, 1172 (1993) and P. L Chisholm et al., xe2x80x9cMonoclonal antibodies to the integrin a-4 subunit inhibit the murine contact hypersensitivity response.xe2x80x9d Eur. J. imunol., 23, 682 (1993)); viii) acute neurotoxic nephritis (see M. S. Mulligan et al., xe2x80x9cRequirements for leukocyte adhesion molecules in nephrotoxic nephritis.xe2x80x9d, J. Clin. Invest., 91, 577 (1993)); ix) tumor metastasis (for examples, see M. Edward, xe2x80x9cintegrins and other adhesion molecules involved in melanocytic tumor progression.xe2x80x9d, Curr. Opin. Oncol., 7, 185 (1995)); x) experimental autoimmune thyroiditis (see R. W. McMurray et al., xe2x80x9cThe role of xcex14 integrin and intercellular adhesion molecule-1 (ICAM-1) in murine experimental autoimmune thyroiditis.xe2x80x9d Autoimmunity, 23, 9 (1996); and xi) ischemic tissue damage following arterial occlusion in rats (see F. Squadrito et al., xe2x80x9cLeukocyte integrin very late antigen-4/vascular cell adhesion molecule-1 adhesion pathway in splanchnic artery occlusion shock.xe2x80x9d Eur. J. Pharmacol., 318, 153 (1996; xii) inhibition of TH2 T-cell cytokine production including IL-4 and IL-5 by VLA-4 antibodies which would attenuate allergic responses (J.Clinical Investigation 100, 3083 (1997). The primary mechanism of action of such antibodies appears to be the inhibition of lymphocyte and monocyte interactions with CAMs associated with components of the extracellular matrix, thereby limiting leukocyte migration to extravascular sites of injury or inflammation and/or limiting the priming and/or activation of leukocytes.
There is additional evidence supporting a possible role for VLA-4 interactions in other diseases, including rheumatoid arthritis; various melanomas, carcinomas, and sarcomas, including multiple myeloma; inflammatory lung disorders; acute respiratory distress syndrome (ARDS); pulmonary fibrosis; atherosclerotic plaque formation; restenosis; uveitis; and circulatory shock (for examples, see A. A. Postigo et al., xe2x80x9cThe xcex14xcex21/VCAM-1 adhesion pathway in physiology and disease.xe2x80x9d, Res. Immunol., 144, 723 (1994) and J.-X. Gao and A. C. Issekutz, xe2x80x9cExpression of VCAM-1 and VLA-4 dependent T-lymphocyte adhesion to dermal fibroblasts stimulated with proinflammatory cytokines.xe2x80x9d Immunol. 89, 375 (1996)).
At present, there is a humanized monoclonal antibody (Antegren(copyright), Athena Neurosciences/Elan) against VLA-4 in clinical development for the treatment of xe2x80x9cflaresxe2x80x9d associated with multiple sclerosis and a humanized monoclonal antibody (ACT-1(copyright)/LDP-02 LeukoSite) against xcex14xcex27 in clinical development for the treatment of inflammatory bowel disease. Several antagonists of VLA-4 and xcex14xcex27 have been described (D. Y. Jackson et al., xe2x80x9cPotent xcex14xcex21 peptide antagonists as potential anti-inflammatory agentsxe2x80x9d, J. Med. Chem., 40, 3359 (1997); H. N. Shroff et al., xe2x80x9cSmall peptide inhibitors of xcex14xcex27 mediated MadCAM-1 adhesion to lymphocytesxe2x80x9d, Bioorg. Med. Chem. Lett., 6, 2495 (1996); K C. Lin et al., xe2x80x9cSelective, tight-binding inhibitors of integrin xcex14xcex21 that inhibit allergic airway responsesxe2x80x9d. J. Med. Chem., 42, 920 (1999); U.S. Pat. No. 5,510,332, WO97103094, WO97/02289, WO96/40781, WO96122966, WO96/20216, WO96/01644, WO96/06108, WO95/15973). There are reports of nonpeptidyl inhibitors of the ligands for xcex14-integrins (WO99/36393, WO98/58902, WO96/31206); A. J. Soures et al., Bioorg. Med. Chem. Lett., 8, 2297 (1998). There still remains a need for low molecular weight, specific inhibitors of VLA-4- and xcex14xcex27-dependent cell adhesion that have improved pharmacokinetic and pharmacodynamic properties such as oral bioavailability and significant duration of action. Such compounds would prove to be useful for the treatment, prevention or suppression of various pathologies mediated by VLA-4 and xcex14xcex27 binding and cell adhesion and activation.
The present invention provides novel compounds of Formula I 
or a pharmaceutically acceptable salt thereof wherein:
R1 and R2 are independently selected from hydrogen, C1-10alkyl, C2-10alkenyl, C2-10alkynyl, Cy and Cy C1-10alkyl, wherein alkyl, alkenyl, alkynyl and Cy are optionally substituted with one to four substituents independently selected from Rb, provided that R1 and R2 are not both hydrogen; or
R1 and R2 together with the nitrogen to which they are attached form a mono- or bicyclic heterocycle each ring having 4 to 7 members and containing 0-2 additional heteroatoms independently selected from oxygen, sulfur and nitrogen, said heterocycle being optionally (1) fused to a benzene ring, or (2) spiro-fused to a 5- or 6-membered saturated or unsaturated ring optionally containing one heteroatom selected from O, S, and N and optionally fused to a benzene ring; wherein said heterocycle, benzene and spiro-fused ring are each optionally substituted with one to four groups selected from Rb;
R3 is
1) hydrogen,
2) C1-10alkyl,
3) C2-10alkenyl,
4) C2-10alkynyl
5) cycloalkyl,
6) heterocyclyl,
7) aryl,
8) heteroaryl,
wherein alkyl, alkenyl, alkynyl, cycloalkyl and heterocyclyl are optionally substituted with one to four substituents selected from Ra, and aryl and heteroaryl are optionally substituted with one to four substituents independently selected from Rb; or
R2 and R3 together with the atoms to which they are attached form a heterocyclic ring of 5 to 7 members with the proviso that R1 and R2 do not form a ring;
R4 is
1) C1-10alkyl,
2) aryl,
3) aryl-C1-10alkyl,
4) biaryl,
5) biaryl-C1-10alkyl,
6) heteroaryl,
7) heteroaryl-C1-10alkyl,
8) heteroaryl-aryl,
9) heteroaryl-aryl-C1-10alkyl,
10) aryl-beteroaryl,
11) aryl-heteroaryl-C1-10alkyl,
wherein the alkyl group is optionally substituted with one to four substituents selected from Ra, and the aryl, biaryl, and heteroaryl groups are substituted with one to four substituents independently selected from Rb,
R5 is
1) hydrogen,
2) C1-10alkyl,
3) C2-10alkenyl,
4) C2-10alkynyl,
wherein alkyl, alkenyl and alkynyl are optionally substituted with one to four substituents independently selected from Ra;
R6 is
1) OH,
2) C1-10alkoxy,
3) C2-10alkenoxy,
4) C2-10alkynoxy,
5) ayloxy,
6) aryl-C1-10alkoxy,
7) NRdRe,
wherein alkyl, alkenyl and alkynyl are optionally substituted with one to four substituents selected from Ra, and aryl is optionally substituted with one to four substituents independently selected from Rb;
R7 is
1) hydrogen,
2) C1-10alkyl,
3) C2-10alkenyl,
4) C2-10alkynyl,
5) ayl,
6) aryl-C1-10alkyl,
wherein alkyl, alkenyl and alkynyl are optionally substituted with one to four substituents selected from Ra, and aryl is optionally substituted with one to four substituents independently selected from Ra;
R8 is
1) hydrogen,
2) C1-10alkyl,
3) C2-10alkenyl,
4) C2-10alkynyl,
5) aryl,
6) heteroaryl,
7) aryl C1-10alkyl,
8) heteroaryl C1-10alkyl,
9) xe2x80x94ORd,
10) xe2x80x94O(CRfRg)nNRdRe,
11) xe2x80x94OC(O)Rd,
12) xe2x80x94OC(O)NRdRe,
13) halogen,
14) xe2x80x94SRd,
15) xe2x80x94S(O)mRd,
16) xe2x80x94S(O)2ORd,
17) xe2x80x94S(O)mNRdRe,
18) xe2x80x94NO2,
19) xe2x80x94NRdRe,
20) xe2x80x94NRdC(O)Re,
21) xe2x80x94NRdS(O)mRe,
22) xe2x80x94NRdC(O)ORe, or
23) xe2x80x94NRdC(O)NRdRe,
wherein alkyl, alkenyl, alkynyl, aryl, heteroaryl are optionally substituted with one to four substituents selected from a group independently selected from Rc;
Rais
1) hydrogen,
2) xe2x80x94ORd,
3) xe2x80x94NO2,
4) halogen
5) xe2x80x94S(O)mRd,
6) xe2x80x94SRd,
7) xe2x80x94S(O)2ORd,
8) xe2x80x94S(O)mNRdRe,
9) xe2x80x94NRdRe,
10) xe2x80x94O(CRfRg)nNRdRe,
11) xe2x80x94C(O)Rd,
12) xe2x80x94CO2Rd,
13) xe2x80x94CO2(CRfRg)nCONRdRe,
14) xe2x80x94OC(O)Rd,
15) xe2x80x94CN,
16) xe2x80x94C(O)NRdRe,
17) xe2x80x94NRdC(O)Re,
18) xe2x80x94OC(O)NRdRe,
19) xe2x80x94NRdC(O)ORe,
20) xe2x80x94NRdC(O)NRdRe,
21) xe2x80x94CRd(Nxe2x80x94ORe),
22) CF3; or
23) xe2x80x94OCF3;
Rb is
1) a group selected from Ra,
2) C1-10alkyl,
3) C2-10alkenyl,
4) C2-10alkynyl,
5) Cy;
6) Cy-C1-10alkyl,
wherein alkyl, alkenyl, alkynyl, and Cy are optionally substituted with one to four substituents selected from a group independently selected from Rc;
Rc is
1) halogen,
2) NRfRg,
3) carboxy,
4) C1-4alkyl optionally substituted with C(O)O-C1-4alkoxy,
5) C1-4alkoxy,
6) aryl,
7) aryl C1-4alkyl,
8) hydroxy,
9) CF3, or
8) aryloxy; or
two Rc groups on adjacent atoms of a benzene ring together form methylenedioxy; Rd and Re are independently selected from hydrogen, C1-10alkyl, C2-10alkenyl, C2-10alkynyl, Cy and Cy C1-10alkyl, wherein alkyl, alkenyl, alkynyl and Cy are optionally substituted with one to four substituents independently selected from Rc; or
Rd and Re together with the atom(s) to which they are attached form a heterocyclic ring of 4 to 7 members containing 0-2 additional heteroatoms independently selected from oxygen, sulfur and nitrogen;
Rf and Rg are independently selected from hydrogen, C1-10alkyl, Cy and Cy-C1-10alkyl; or
Rf and Rg together with the carbon to which they are attached form a ring of 5 to 7 members containing 0-2 heteroatoms independently selected from oxygen, sulfur and nitrogen;
Cy is independently selected from cycloalkyl, heterocyclyl, aryl, or heteroaryl;
m is an integer from 1 to 2;
n is an integer from 1 to 10;
Y is
1) a bond, or
2) xe2x80x94C(R7)(R8)xe2x80x94
In one embodiment of compounds of formula I, R1 is hydrogen or C1-5alkyl, and R2 is selected from Cy and Cy-C1-5=l alkly wherein Cy is optionally substituted with one to four substituents independently selected from Rb.
In another embodiment of compounds of formula I, R1 and R2 together with the nitrogen to which they are attached form a mono- or bicyclic heterocycle each ring having 4 to 7 members and containing 0-2 additional heteroatoms independently selected from oxygen, sulfur and nitrogen, said heterocycle being optionally (1) fused to a benzene ring, or (2) spiro-fused to a 5- or 6-membered saturated or unsaturated ring optionally containing one heteroatom selected from O, S, and N and optionally fused to a benzene zing; wherein said heterocycle, benzene and spiro-fused ring are each optionally substituted with one to four groups selected from Rb. Examples of ring systems (which may be optionally substituted as described above) include pyrrolidine, morpholine, piperidine, thiamorpholine, azacycloheptane, tetrahydroquinoline, tetaydroisoquinoline, 2-azabicyclo[3.2.1]octane, 3,4-benzo-2,8-diazaspiro[4.5]decane, 3,4-benzo-8-azaspiro[4.5]decane, 3,4-benzo-8-azaspiro[4.5]dec-1-ene. In a preferred embodiment R1 and R2 together with the nitrogen to which they are attached form a monocyclic heterocycle having 4 to 7 members and containing 0-2 additional heteroatoms independently selected from oxygen, sulfur and nitrogen, said heterocycle being optionally substituted with one to four groups selected from phenyl optionally substituted with halogen or C1-3alkoxy, benzyl, C1-5alkyl optionally substituted with hydroxy or NRfRg, CO2Rd, C(O)Rd, and C(O)NRdRe.
In another embodiment, R3 is hydrogen.
In another embodiment R5 is hydrogen, and R4 is biaryl-C1-5alkyl wherein aryl is optionally substituted with one to four groups selected from Rb. Prefereably R4 is biphenylmethyl optionally substituted with one or two groups selected from Rb. More preferably R4 is biphenylmethyl substituted with one or two groups selected from Rb, and wherein one of the substituents is attached to the 2xe2x80x2-position. Even more preferred, R4 is selected from 2xe2x80x2-cyano-biphenylmethyl, 2xe2x80x2-methoxy-biphenylmethyl and 2xe2x80x2,6xe2x80x2-bis(methoxy)biphenylmethyl.
In another embodiment Y is a bond.
In another embodiment R6 is OH or a pharmaceutically acceptable salt thereof.
Examples of compounds of the present invention include:
The present invention provides, in another aspect, a method for the prevention or treatment of diseases, disorders, conditions or symptoms mediated by cell adhesion in a mammal which comprises administering to said mamal an effective amount of a compound of Formula I. More particularly said disease or disorder is selected from asthma, allergic rhinitis, multiple sclerosis, atherosclerosis, and inflammatory bowel disease.
In another aspect the present invention provides a method for preventing the action of VLA-4 in a mammal which comprises administering to said mammal a thereapeutically effective amount of a compound of formula I.
Another aspect of the present invention provides a pharmaceutical composition which comprises a compound of formula I and a pharmaceutically acceptable carrier.
Unless otherwise specified the following terms have the given meanings:
xe2x80x9cAkylxe2x80x9d, as well as other groups having the prefix xe2x80x9calkxe2x80x9d, such as alkoxy, alkanoyl, means carbon chains which may be linear or branched or combinations thereof. Examples of alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, sec- and tert-butyl, pentyl, hexyl, heptyl, octyl, nonyl, and the like.
xe2x80x9cAlkenylxe2x80x9d means carbon chains which contain at least one carbon-carbon double bond, and which may be linear or branched or combinations thereof. Examples of alkenyl include vinyl, allyl, isopropenyl, pentenyl, hexenyl, heptenyl, 1-propenyl, 2-butenyl, 2-methyl-2-butenyl, and the like.
xe2x80x9cAlkynylxe2x80x9d means carbon chains which contain at least one carbon-carbon triple bond, and which may be linear or branched or combinations thereof. Examples of alkynyl include ethynyl, propargyl, 3-methyl-1-pentynyl, 2-heptynyl and and the like.
xe2x80x9cCycloalkylxe2x80x9d means mono- or bicyclic saturated carbocyclic rings, each of which having from 3 to 10 carbon atoms. The term also includes monocyclic rings fused to an aryl group in which the point of attachment is on the non-aromatic portion. Examples of cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, tetrahydronaphthyl, decahydronaphthyl, indanyl, and the like.
xe2x80x9cArylxe2x80x9d means mono- or bicyclic aromatic rings containing only carbon atoms. The term also includes aryl group fused to a monocyclic cycloalkyl or monocyclic heterocyclyl group in which the point of attachment is on the aromatic portion. Examples of aryl include phenyl, naphthyl, indanyl, indenyl, tetrahydronaphthyl, 2,3-dihydrobenzofuranyl, dihydrobenzopyranyl, 1,4-benzodioxanyl, and the like.
xe2x80x9cHeteroarylxe2x80x9d means a mono- or bicyclic aromatic ring containing at least one heteroatom selected from N, O and S, with each ring containing 5 to 6 atoms. Examples of heteroaryl include pyrrolyl, isoxazolyl, isothiazolyl, pyrazolyl, pyridyl, oxazolyl, oxadiazolyl, thiadiazolyl, thiazolyl, imidazolyl, triazolyl, tetrazolyl, furanyl, triazinyl, thienyl, pyrimidyl, pyridazinyl, pyrazinyl, benzoxazolyl, benzothiazolyl, benzimidazolyl, benzofuranyl, benzothiophenyl, furo(2,3-b)pyridyl, quinolyl, indolyl, isoquinolyl, and the like.
xe2x80x9cHeterocyclylxe2x80x9d means mono- or bicyclic saturated rings containing at least one heteroatom selected from N, S and O, each of said ring having from 3 to 10 atoms in which the point of attachment may be carbon or nitrogen. The term also includes monocyclic heterocycle fused to an aryl or heteroaryl group in which the point of attachment is on the non-aromatic portion. Examples of xe2x80x9cheterocyclylxe2x80x9d include pyrrolidinyl, piperidinyl, piperazinyl, morpholine, thiamorpholine, benzoazacycloheptyl, azacycloheptyl, azabicyclo[3.2.1]octyl, imidazolidinyl, 2,3-dihydrofuro(2,3-b)pyridyl, benzoxazinyl, tetrahydrohydroquinolinyl, tetrahydroisoquinolinyl, dihydroindolyl, and the like. The term also includes partially unsaturated monocyclic rings that are not aromatic, such as 2- or 4-pyridones attached through the nitrogen or N-substituted-(1H,3H)pyximidine-2,4-diones (N-substituted uracils).
xe2x80x9cHalogenxe2x80x9d includes fluorine, chlorine, bromine and iodine.
Optical Isomersxe2x80x94Diastereomersxe2x80x94Geometric Isomersxe2x80x94Tautomers
Compounds of Formula I contain one or more asymmetric centers and can thus occur as racemates and racemic mixtures, single enantiomers, diastereomeric mixtures and individual diastereomers. The present invention is meant to comprehend all such isomeric forms of the compounds of Formula I.
Some of the compounds described herein contain olefinic double bonds, and unless specified otherwise, are meant to include both E and Z geometric isomers.
Some of the compounds described herein may exist with different points of attachment of hydrogen, referred to as tautomers. Such an example may be a ketone and its enol form known as ketonol tautomers. The individual tautomers as well as mixture thereof are encompassed with compounds of Formula I.
Compounds of the Formula I may be separated into diastereoisomeric pairs of enantiomers by, for example, factional crystallization from a suitable solvent, for example methanol or ethyl acetate or a mixture thereof. The pair of enantiomers thus obtained may be separated into individual stereoisomers by conventional means, for example by the use of an optically active acid as a resolving agent.
Alternatively, any enantiomer of a compound of the general Formula I or Ia may be obtained by stereospecific synthesis using optically pure starting materials or reagents of known configuration.
Salts
The term xe2x80x9cpharmaceutically acceptable saltsxe2x80x9d refers to salts prepared from pharmaceutically acceptable non-toxic bases or acids including inorganic or organic bases and inorganic or organic acids. Salts derived from inorganic bases include aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic salts, manganous, potassium, sodium, zinc, and the like. Particularly preferred are the ammonium, calcium, magnesium, potassium, and sodium salts. Salts derived from pharmaceutically acceptable organic non-toxic gases include salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as arginine, betaine, caffeine, choline, N,Nxe2x80x2-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethyl-morpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, and the like.
When the compound of the present invention is basic, salts may be prepared from pharmaceutically acceptable non-toxic acids, including inorganic and organic acids. Such acids include acetic, benzenesulfonic, benzoic, camphorsulfonic, citric, ethanesulfonic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric, pamoic, pantothenic, phosphoric, succinic, sulfuric, tartaric, p-toluenesulfonic acid, and the like. Particularly preferred are citric, hydrobromic, hydrochloric, maleic, phosphoric, sulfuric, and tartaric acids.
It will be understood that, as used herein, references to the compounds of Formula I are meant to also include the pharmaceutically acceptable salts.
Utilities
The ability of the compounds of Fomula I to antagonize the actions of VLA-4 and/or xcex14xcex27 integrin makes them useful for preventing or reversing the symptoms, disorders or diseases induced by the binding of VLA-4 and or xcex14xcex27 to their various respective ligands. Thus, these antagonists will inhibit cell adhesion processes including cell activation, migration, proliferation and differentiation. Accordingly, another aspect of the present invention provides a method for the treatment (including prevention, alleviation, amelioration or suppression) of diseases or disorders or symptoms mediated by VLA-4 and/or xcex14xcex27 binding and cell adhesion and activation, which comprises administering to a mammal an effective amount of a compound of Formula I. Such diseases, disorders, conditions or symptoms are for example (1) multiple sclerosis, (2) asthma, (3) allergic rhinitis, (4) allergic conjunctivitis, (5) inflammatory lung diseases, (6) rheumatoid arthritis, (7) septic arthritis, (8) type I diabetes, (9) organ transplantation rejection, (10) restenosis, (11) autologous bone marrow transplantation, (12) inflammatory sequelae of viral infections, (13) myocarditis, (14) inflammatory bowel disease including ulcerative colitis and Crohn""s disease, (15) certain types of toxic and immune-based nephritis, (16) contact dermal hypersensitivity, (17) psoriasis, (18) tumor metastasis, (19) hepatitis and (20) atherosclerosis.
Dose Ranges
The magnitude of prophylactic or therapeutic dose of a compound of Formula I will, of course, vary with the nature of the severity of the condition to be treated and with the particular compound of Formula I and its route of administration. It will also vary according to the age, weight and response of the individual patient. In general, the daily dose range lie within the range of from about 0.001 mg to about 100 mg per kg body weight of a mammal, preferably 0.01 mg to about 50 mg per kg, and most preferably 0.1 to 10 mg per kg, in single or divided doses. On the other hand, it may be necessary to use dosages outside these limits in some cases.
For use where a composition for intravenous administration is employed, a suitable dosage range is from about 0.001 mg to about 25 mg (preferably from 0.01 mg to about 1 mg) of a compound of Formula I per kg of body weight per day and for cytoprotective use from about 0.1 mg to about 100 mg (preferably from about 1 mg to about 100 mg and more preferably from about 1 mg to about 10 mg) of a compound of Formula I per kg of body weight per day.
In the case where an oral composition is employed, a suitable dosage range is, e.g. from about 0.01 mg to about 100 mg of a compound of Formula I per kg of body weight per day, preferably from about 0.1 mg to about 10 mg per kg and for cytoprotective use from 0.1 mg to about 100 mg (preferably from about 1 mg to about 100 mg and more preferably from about 10 mg to about 100 mg) of a compound of Formula I per kg of body weight per day.
For the treatment of diseases of the eye, ophthalmic preparations for ocular administration comprising 0.001-1% by weight solutions or suspensions of the compounds of Formula I in an acceptable ophthalmic formulation may be used.
Pharmaceutical Compositions
Another aspect of the present invention provides pharmaceutical compositions which comprises a compound of Formula I and a pharmaceutically acceptable carrier. The term xe2x80x9ccompositionxe2x80x9d, as in pharmaceutical composition, is intended to encompass a product comprising the active ingredient(s), and the inert ingredient(s) (pharmaceutically acceptable excipients) that make up the carrier, as well as any product which results, directly or indirectly, from combination, complexation or aggregation of any two or more of the ingredients, or from dissociation of one or more of the ingredients, or from other types of reactions or interactions of one or more of the ingredients. Accordingly, the pharmaceutical compositions of the present invention encompass any composition made by admixing a compound of Formula I, additional active ingredient(s), and pharmaceutically acceptable excipients.
Any suitable route of administration may be employed for providing a mammal, especially a human with an effective dosage of a compound of the present invention. For example, oral, rectal, topical, parenteral, ocular, pulmonary, nasal, and the like may be employed. Dosage forms include tablets, troches, dispersions, suspensions, solutions, capsules, creams, ointments, aerosols, and the like.
The pharmaceutical compositions of the present invention comprise a compound of Formula I as an active ingredient or a pharmaceutically acceptable salt thereof, and may also contain a pharmaceutically acceptable carrier and optionally other therapeutic ingredients. The term xe2x80x9cpharmaceutically acceptable saltsxe2x80x9d refers to salts prepared from pharmaceutically acceptable non-toxic bases or acids including inorganic bases or acids and organic bases or acids.
The compositions include compositions suitable for oral, rectal, topical, parenteral (including subcutaneous, intramuscular, and intravenous), ocular (ophthalmic), pulmonary (aerosol inhalation), or nasal administration, although the most suitable route in any given case will depend on the nature and severity of the conditions being treated and on the nature of the active ingredient. They may be conveniently presented in unit dosage form and prepared by any of the methods well-known in the art of pharmacy.
For administration by inhalation, the compounds of the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or nebulisers. The compounds may also be delivered as powders which may be formulated and the powder composition may be inhaled with the aid of an insufflation powder inhaler device. The preferred delivery systems for inhalation are metered dose inhalation (MDI) aerosol, which may be formulated as a suspension or solution of a compound of Formula I in suitable propellants, such as fluorocarbons or hydrocarbons and dry powder inhalation (DPI) aerosol, which may be formulated as a dry powder of a compound of Formula I with or without additional excipients.
Suitable topical formulations of a compound of formula I include transdermal devices, aerosols, creams, ointments, lotions, dusting powders, and the like.
In practical use, the compounds of Formula I can be combined as the active ingredient in intimate admixture with a pharmaceutical carrier according to conventional pharmaceutical compounding techniques. The carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g., oral or parenteral (including intravenous). In preparing the compositions for oral dosage form, any of the usual pharmaceutical media may be employed, such as, for example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents and the like in the case of oral liquid preparations, such as, for example, suspensions, elixirs and solutions; or carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents and the like in the case of oral solid preparations such as, for example, powders, capsules and tablets, with the solid oral preparations being preferred over the liquid preparations. Because of their ease of administration, tablets and capsules represent the most advantageous oral dosage unit form in which case solid pharmaceutical carriers are obviously employed. If desired, tablets may be coated by standard aqueous or nonaqueous techniques.
In addition to the common dosage forms set out above, the compounds of Formula I may also be administered by controlled release means and/or delivery devices such as those described in U.S. Pat. Nos. 3,845,770; 3,916,899; 3,536,809; 3,598,123; 3,630,200 and 4,008,719.
Pharmaceutical compositions of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient, as a powder or granules or as a solution or a suspension in an aqueous liquid, a non-aqueous liquid, an oil-in-water emulsion or a water-in-oil liquid emulsion. Such compositions may be prepared by any of the methods of pharmacy but all methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more necessary ingredients. In general, the compositions are prepared by uniformly and intimately admixing the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the desired presentation. For example, a tablet may be prepared by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine, the active ingredient in a free-flowing form such as powder or granules, optionally mixed with a binder, lubricant, inert diluent, surface active or dispersing agent. Molded tablets may be made by molding in a suitable machine, a mixture of the powdered compound moistened with an inert liquid diluent. Desirably, each tablet contains from about 1 mg to about 500 mg of the active ingredient and each cachet or capsule contains from about 1 to about 500 mg of the active ingredient.
The following are examples of representative pharmaceutical dosage forms for the compounds of Formula I:
Combination Therapy
Compounds of Formula I may be used in combination with other drugs that are used in the treatment/prevention/suppression or amelioration of the diseases or conditions for which compounds of Formula I are useful. Such other drugs may be administered, by a route and in an amount commonly used therefor, contemporaneously or sequentially with a compound of Formula I. When a compound of Formula I is used contemporaneously with one or more other drugs, a pharmaceutical composition containing such other drugs in addition to the compound of Formula I is preferred. Accordingly, the pharmaceutical compositions of the present invention include those that also contain one or more other active ingredients, in addition to a compound of Formula I. Examples of other active ingredients that may be combined with a compound of Formula I, either administered separately or in the same pharmaceutical compositions, include, but are not limited to: (a) other VLA-4 antagonists such as those described in U.S. Pat. No. 5,510,332, WO97/03094, WO97/02289, WO96/40781, WO96/22966, WO96/20216, WO96/01644, WO96/06108, WO95/15973 and WO96/31206; (b) steroids such as beclomethasone, methylprednisolone, betamethasone, prednisone, dexamethasone, and hydrocortisone; (c) immunosuppressants such as cyclosporin, tacrolimus, rapamycin and other FK-506 type immunosuppressants; (d) antihistamines (H1-histamine antagonists) such as bromopheniramine, chlorphenirarnine, dexchlorpheniramine, triprolidine, clemastine, diphenhydramine, diphenylpyraline, tripelennamine, hydroxyzine, methdilazine, promethazine, trimeprazine, azatadine, cyproheptadine, antazoline, pheniramine pyrilamine, astemizole, terfenadine, loratadine, cetirizine, fexofenadine, descarboethoxyloratadine, and the like; (e) non-steroidal anti-asthmatics such as b2-agonists (terbutaline, metaproterenol, fenoterol, isoetharine, albuterol, bitolterol, salmeterol and pirbuterol), theophylline, cromolyn sodium, atropine, ipratropium bromide, leukotriene antagonists (zafirlukast, montelukast, pranlukatst, iralukast, pobilukast, SKB-106,203), leukotriene biosynthesis inhibitors (zileuton, BAY-1005); (f) non-steroidal antiinflammatory agents (NSAIDs) such as propionic acid derivatives (alrinoprofen, benoxaprofen, bucloxic acid, carprofen, fenbufen, fenoprofen, fluprofen, flurbiprofen, ibuprofen, indoprofen, ketprofen, miroprofen, naproxen, oxaprozin, pirprofen, pranoprofen, suprofen, tiaprofenic acid, and tioxaprofen), acetic acid derivatives (indomethacin, acemetacin, alclofenac, clidanac, diclofenac, fenclofenac, fenclozic acid, fentiazac, furofenac, ibufenac, isoxepac, oxpinac, sulindac, tiopinac, tolmetin, zidometacin, and zomepirac), fenamic acid derivatives (flufenamic acid, meclofenamic acid, mefenamic acid, niflumic acid and tolfenamic acid), biphenylcarboxylic acid derivatives (diflunisal and flufenisal), oxicams (isoxicam, piroxicam, sudoxicam and tenoxican), salicylates (acetyl salicylic acid, sulfasalazine) and the pyrazolones (apazone, bezpiperylon, feprazone, mofebutazone, oxyphenbutazone, phenylbutazone); (g) cyclooxygenase-2 (COX-2) inhibitors such as celecoxib; (h) inhibitors of phosphodiesterase type IV (PDE-IV); (i) antagonists of the chemokine receptors, especially CCR-1, CCR-2, and CCR-3; (j) cholesterol lowering agents such as HMG-CoA reductase inhibitors (lovastatin, simvastatin and pravastatin, fluvastatin, atorvastatin, and other statins), sequestrants (cholestyramine and colestipol), nicotinic acid, fenofibric acid derivatives (gemfibrozil, clofibrat, fenofibrate and benzafibrate), and probucol; (k) anti-diabetic agents such as insulin, sulfonylureas, biguanides (metformin), a-glucosidase inhibitors (acarbose) and glitazones (troglitazone, pioglitazone, englitazone, MCC-555, BRL49653 and the like); (l) preparations of interferon beta (interferon beta-1a, interferon beta-1b); (m) anticholinergic agents such as muscarinic antagonists (ipratropium bromide); (n) other compounds such as 5-aminosalicylic acid and prodrugs thereof, antimetabolites such as azathioprine and 6-mercaptopurine, and cytotoxic cancer chemotherapeutic agents.
The weight ratio of the compound of the Formula I to the second active ingredient may be varied and will depend upon the effective dose of each ingredient. Generally, an effective dose of each will be used. Thus, for example, when a compound of the Formula I is combined with an NSAID the weight ratio of the compound of the Formula I to the NSAID will generally range from about 1000:1 to about 1:1000, preferably about 200:1 to about 1:200. Combinations of a compound of the Formula I and other active ingredients will generally also be within the aforementioned range, but in each case, an effective dose of each active ingredient should be used.
Compounds of the present invention may be prepared by procedures illustrated in the accompanying schemes. In the first method (Scheme 1), a resin-based synthetic strategy is outlined where the resin employed is represented by the ball (). An N-Fmoc-protected amino acid derivative A (Fmoc=fluorenylmethoxycarbonyl) is loaded on to the appropriate hydroxyl-containing resin (the choice of resin being dependent on type of linker used, in this case Wang resin was utilized) using 1-ethyl-3-(3xe2x80x2-dimethylaminopropyl)carbodiimide (EDC) and 4-dimethylaminopyridine (4-DMAP) in a solvent such as methylene chloride and teterahydrofuran or dimethylformamide (DMF) to give B. The Fmoc protecting group is removed with piperidine in DMF to yield free amine C. A reactive carbonyl reagent such as 4-nitrophenylchoroformate in the presence of a base such as diisopropylethylaniine is added to a slurry of C and, following washing, is reacted with an amine D to form urea E. Alternatively, C may be reacted with an isocyante reagent F to form E directly. The final product is removed from the resin with strong acid (in this instance, trifluoroacetic acid to yield compounds of the present invention G. 
In the second method (Scheme 2), standard solution phase synthetic methodology is outlined. Many amino acid derivatives are commercially available as the t-butyl or methyl esters and may be used directly in the synthesis outlined below. Amino acid t-butyl esters A may be prepared from amino acids directly by treament with isobutylene and sulfuric acid in diglyme or dioxane. Alternatively, N-Boc-protected amino acid derivative is treated with tert-butyl 2,2,2-trichloroacetimidate in the presence of boron trifluoride etherate followed by treatment with strong acid (HCl in ethyl acetate or sulfuric acid in t-butyl acetate) to remove the t-BOC group to yield tert-butyl ester A. A reactive carbonyl reagent such as 4-nitrophenylchoroformate in the presence of a base such as diisopropylethylamine is added to a solution of A which is subsequently reacted with an amine B to form urea D. Alternatively, A may be reacted with an isocyante reagent C to form D directly. The final product is hydrolyzed with strong acid (in this instance, trifluoroacetic acid) if D is a tert-butyl ester or hydroxide if D is a methyl ester to yield compounds of the present invention E. 
In a third method (Scheme 3), a late stage intermediate aryl bromide or iodide is coupled to an appropriately substituted aryl or heteroaryl boronic acid to give a subset of compounds of the present invention (R4=biaryl-substituted-alkyl or heteroaryl-aryl-substituted-alkyl, R3=hydrogen). For example, 4-iodo or 4-bromophenyl-derivative A is converted to the t-butyl ester B by treatment with isobutylene and sulfuric acid. Alternatively the N-Boc-4-iodo- or 4-bromo-phenyl-denivative C is reacted with tert-butyl 2,2,2-trichloroacetimidate in the presence of boron trifluoride etherate in methylene chloride-cyclohexane followed by treatment with strong acid 
(HCl in ethyl acetate or sulfuric acid in t-butyl acetate) to remove the t-BOC group to yield tert-butyl ester B which is subsequently reacted with p-nitrophenylchloroformate and amine D or directly with isocyanate E as described above to yield urea F. Substituted aryl or heteroaryl boronic acids are coupled to F in the presence of a palladium(0) reagent, such as tetrakis(triphenylphosphine)palladium under Suzuki conditions (N. Miyaura et al., Synth. Commun., 1981, 11, 513-519) to yield G. The tert-butyl ester is then removed by treatment with strong acid (TFA) to yield the desired product H. If the aryl or heteroaryl boronic acid is not commercially available, but the corresponding bromide or iodide is, then the bromide or iodide can be converted into the desired boronic acid by treatment with an alkyllithium reagent in tetrahydrofuran at low temperature followed by addition of trimethyl or triisopropyl borate. Hydrolysis to the boronic acid can be effected by treatment of the intermediate with aqueous base and then acid.
Alternatively, the aryl coupling reaction may be performed by application of Stille-type carbon-carbon bond forming conditions (Scheme 4). (A. M. Echavarren and J. K. Stille, J. Am. Chem. Soc. 1987, 109, 5478-5486). The aryl bromide or iodide intermediate A (described in Scheme 3) is converted into its trialkyltin derivative B using hexamethylditin in the presence of a palladium(0) catalyst and lithium chloride and then reacted with an appropriately substituted aryl or heteroaryl bromide, iodide, or triflate in the presence of a palladium reagent, such as tetrakis(triphenylphosphine)palladium(0) or tris(dibenzylideneacetone)dipalladium(0), in a suitable solvent, such as toluene, dioxane, DMF, or 1-methyl-2-pyrrolidinone, to give intermediate C. The tert-butyl ester is then removed by treatment with strong acid (TFA) to yield the desired product D. Biphenyl amino acids suitable for attachment to resin (D where R1 is fluorenylmethyloxy) may be prepared by this route as well. Superior coupling conversions and rates may be elicited by application of the method of Farina (J. Org. Chem. 5434, 1993) 
Compounds wherein the middle ring is heteroaryl (E) may be prepared (Scheme 5) in a similar fashion starting from the appropriate heteroaryl bromide or iodide D using Suzuki-type conditions as depicted in Scheme 3 or from the corresponding heteroaryl trimethyltin using Stille-type conditions as depicted in Scheme 4. The requisite heteroaryl halides D may be prepared via conventional electrophilic halogenation of the N-Boc-heteroaryl-alanine tert-butyl ester interrmediate B. B may be prepared from the known aliphatic iodo intermediate A in carbon-carbon bond formation using zinc/copper couple and palladium(II) (M. J. Dunn et al., SYNLETT 1993, 499-500). 
N-FMOC-(L)-4-(2xe2x80x2-cyanophenyl)phenylalanine
Step A
N-FMOC-(L)-4-iodophenylalanine, t-butyl Ester
To a solution of 15 g (51 mmol) of (L)-4-iodo-phenylalanine in 100 ml of diglyme and 15 ml of concentrated H2SO4 was added 30 ml of condensed isobutylene. The vessel was agitated overnight and the crude product was diluted with 100 ml of ethyl acetate. The solution was added to excess sodium hydroxide solution while maintaining the temperature below 30xc2x0 C. A white precipitate formed which dissolved upon addition of sodium hydroxide solution. The resulting mixture was filtered and the aqueous phase was extracted with ethyl acetate. The combined extracts were washed with brine and dried over anhydrous magnesium sulfate. The mixture was filtered and concentrated in vacuo to give a solution of the product in diglyme. The solution was diluted with 200 ml of ether and was treated with excess 1N HCl in ether with rapid stirring. The resulting precipitate was collected and dried in vacuo after washing with ether. A white solid (9.01 g) was collected of 4-iodophenylalanine t-butyl ester hydrochloride. To a suspension of 5.1 g (13.3 mmol) of the amnine hydrochloride in 30 ml of methylene chloride was added 3.6 g (27 mmol) of diisopropyl ethyl amine followed by 3.43 g (0.013 g) of FMOCCl. The solution was stirred overnight at room temperature, washed with 1N HCl solution (3xc3x9750 ml), water (1xc3x9750 ml), saturated sodium carbonate solution (2xc3x9750 ml) and brine (1xc3x9750 ml). The solution was dried over MgSO4, filtered and concentrated in vacuo to give 6.43 g of N-FMOC-(L)-4-iodophenylalanine, t-butyl ester as a white foam.
300 MHz 1H NMR (CDCl3): d 1.44 (s, 9H); 3.05 (d, 2H);4.20-4.60 (m, 4H); 5.30 (m, 1H); 6.90 (d, 2H), 7.30-7.80 (m, 12H).
Step B
N-FMOC-(L)-4-trimethylstannyl-phenylalanine, tert-butyl Ester
In a dry 250 ml round bottom flask was added 6.20 g (10.5 mmol) of the product of Step A, 0.48 g (115 mmol) LiCl and 0.6 g (0.52 mmol) of palladium tetrakistriphenylphosphine followed by 50 ml of dry dioxane. The mixture was stirred for 5 minutes. 5.2 g (15.8 mmol) of hexamethylditin was added and the reaction mixture was degassed and then heated at 90xc2x0 C. The reaction mixture gave a black suspension after 15 minutes. Completion of the conversion was determined by TLC (10% EtOAc/hexanes; sm r.f.=0.3, product r.f.=0.4). The mixture was diluted with 100 ml of hexanes and stirred to give a precipitate. The suspension was filtered through celite and concentrated in vacuo to give a gum. The residue was purified by flash chromatography over silica gel eluting with 10% EtOAc/hexanes to give 5.02 g of the stannane (77% yield).
300 MHz 1H NMR (CDCl3): d 0.30 (s, 9H); 1.45 (s, 9H); 3.20 (d, 2H), 4.20-4.60 (m, 4H); 5.29 (d, 1H); 7.12 (d, 2H); 7.22-7.45 (m, 6H); 7.59 (d, 2H), 7.75 (d, 2H).
Step C
N-FMOC-(L)-4-(2xe2x80x2-cyanophenyl)phenylalanine, tert-butyl Ester
In a clean, dry round bottom flask fitted with a reflux condenser vented through a three way valve attached to a vacuum source and nitrogen gas was added 1.56 g (6.8 mmol) of 2-iodobenzonitrile, 0.117 (0.12 mmol) of tris(dibenzylidineacetone)-dipalladium (0), 0.8 g (19 mmol) of LiCl and 0.15 g (0.5 mmol) of triphenylarsine followed by 30 ml of N-methylpyrrolidinone (NMP). The mixture was degassed and stirred for 10 minutes at which time most of the catalyst mixture had dissolved. 3.9 g (6.21 mmol) of the product of Step B was added in 10 ml of NMP and the reaction was heated to 80xc2x0 C. for 90 minutes. TLC (10% EtOAc/hexanes) indicated complete consumption of stannane (rf=0.4) and formation of the desired product (rf=0.1). The solution was cooled to room temperature and diluted with 50 ml of EtOAc. The solution was stirred with 20 ml of saturated KF for 20 minutes. The mixture was diluted with 200 ml of EtOAc and washed with water (6xc3x9775 ml), brine (1xc3x9750 ml) and was dried over MgSO4. The mixture was filtered and concentrated in vacuo and the residue was purified by Biotage Flash chromatography over silica gel eluting with 20% EtOAc/hexanes to give 1.91 g (54% yield) of the title compound.
300 MHz 1H NMR (CDCl3): d 1.45 (s, 9H); 3.19 (d, 2H); 4.20-4.68 (m, 4H); 5.40 (d, 1H); 7.25-7.55 (m, 12H); 7.65 (m, 2H), 7.80 (d, 2H).
Step D
N-FMOC-(L)-4-(2xe2x80x2-cyanophenylphenylalanine
2.4 g of the product of Step C was treated with 50 ml of a mixture of 50% trifluoroacetic acid in methylene chloride. The reaction mixture was concentrated in vacuo. The residue was azeotropically dried by concentration from toluene to give the desired product as a foam.
300 MHz 1H NMR (CD3OD): d 3.02 (dd, 1H); 3.30 (dd, 1H); 4.05-4.35 (m, 3H); 4.52 (m, 1H); 7.10-7.50 (m, 12H); 7.60 (m, 2H), 7.78 (d, 2H).
N-(FMOC)-(L)-4-(2xe2x80x2-methoxyphenyl)phenylalanine
Step A
N-(Boc)-(L)-4-iodphenylalanine, tert-butyl Ester
To a suspension of 7.5 g (0.019 m) of 4-iodophenylalanine t-butyl ester (Reference Example 1 Step A prior to treatment with HCl) in 100 ml of dichloromethane was added 2.52 g 0.019 m of diisopropyl ethyl amine followed by 4.14 g of ditertbutyldicarbonate. The reaction mixture was stirred over night at room temperature, washed with 1N HCl (2xc3x9725 ml), water (2xc3x9725 ml), saturated NaHCO3 (1xc3x9725 ml), brine (1xc3x9725 ml) and was dried over MgSO4. The mixture was filtered and concentrated in vacuo to to give the desired product as a gum 8.8 g (100% yield).
300 MHz 1H NMR (CDCl3): xcex41.39 (s, 18H); 2.98 (AB, 2H); 4.4 (dd, 2H); 5.0 bd, 1H); 6.92 (d, 2H); 7.62 (d, 2H).
Step B
N-(Boc)-(L)-4-(2xe2x80x2-methoxyphenyl)phenylalanine, tert-butyl Ester
7.97 g (0.018 m) of the product of Step A was dissolved in 160 ml of 2:1 toluene:ethanol. To this solution was added 2.99 g (0.0198 m) 2-methoxyphenylboronic acid, 0.69 g of tetrakistriphenylphosphine palladium (0) and 22.7 ml (0.45 m) of 2.0 M sodium carbonate in water. The reaction mixture was degassed three times and then heated at 90xc2x0 C. for 90 minutes at which time the reaction mixture was black. The mixture was diluted with 300 ml of ethyl acetate and was washed with water (3xc3x97150 ml) and brine (2xc3x97100 ml) and was dried over MgSO4. The mixture was filtered and concentrated in vacuo. The residue was purified by flash chromatography over silica gel eluting with 10% EtOAc/hexanes to give 6.89 g (88% yield) of the desired product as a white solid.
300 MHz 1H NMR (CDCl3): xcex41.45 (s, 18H); 3.10 (d, 2H); 3.80 (s, 3H); 4.5 (dd, 2H); 5.1 bd, 1H); 7.0 (m, 2H); 7.22 (d, 2H); 7.30 (d, 2H); 7.49 (d, 2H); 7.62 (d, 2H).
Step C
N-(FMOC)-(L)-4-(2xe2x80x2-methoxyphenyl)phenylalanine
To a solution of 4.85 g (0.0113 m) of the product of Step B in 100 ml of t-butyl acetate was added 5.53 g (0.056 m) of concentrated sulfuric acid. The solution was stirred at room temperature for 2 hours and then carefully neutralized by addition of saturated aqueous NaHCO3 solution. The solution was washed with NaHCO3 solution, dried over NaSO4, filtered and concentrated in vacuo. To a solution of 4.42 g of amine in 150 ml of methylene chloride was added at 0xc2x0 C. 1.74 g (13.5 mmol) of diisopropylethyl amine followed by 3.48 g (13.5 mmol) of FMOCCl. The solution was stirred for 2 hours and washed with 1N HCl (3xc3x9750 ml), saturated NaHCO3 solution (2xc3x9750 ml) and brine (1xc3x9750 ml). The mixture was filtered and concentrated in vacuo. The residue was purified by flash chromatography over silica gel eluting with a gradient of 10-25% EtOAc/hexanes to give 7.10 g (88% yield) of the desired product as a glass. The material was dissolved in 125 ml of 50% trifluoracetic acid/methylene chloride and stirred at room temperature for 2.5 hours. The solution was concentrated in vacuo and the residue was redissolved in toluene and concentrated in vacuo to give 7.01 g of the desired product. 96% pure by HPLC (254 nm).
300 MHz 1H NMR (CDCl3): xcex43.20 (m, 2H); 3.76 (s, 3H); 4.21 (t, 1H); 4.41 (m, 4H); 4.76 (dd, 1H); 5.32 (d, 1H); 6.8-7.8 (m, 16H).
N-(FMOC)-(L)-4-(1-pyrrolidino-carbonyloxy)phenylalanine
Step A
N-(Boc)-(L)-tyrosine, tert-butyl Ester
To a solution of 9.82 g (0.041 m) of (L)-tyrosine, tert-butyl ester in 150 ml of methylene chloride and 20 ml of DMF was added 5.2 g (0.04 m) of triethyl amine followed by 9.03 g (0.04 m) of ditertbutyldicarbonate. The reaction mixture was stirred for 2 hours at room temperature and was then washed with 1 N HCl (3xc3x9750 ml), NaHCO3 solution (1xc3x9750 ml) and brine (1xc3x9750 ml) and was dried over MgSO4. The mixture was filtered and concentrated in vacuo to give 13.59 g (98% yield) of a white solid.
300 MHz 1H NMR (CDCl3): 1.42 (s, 18H); 2.95 (d, 2H); 4.39 (dd, 1H); 5.01 (d, 1H); 6.15 (s, 1H); 6.70 (d, 2H); 7.00 d, 2H).
Step B
N-(Boc)-(L)-4-(1-pyrrolidino-carbonyloxy)phenylalanine, tert-butyl Ester
To a solution of 8.18 g (0.024 m) of the product of Step A in a clean, dry flask dissolved in 100 ml of THF under a dry nitrogen atmosphere was added at 0xc2x0 C. 25.5 ml (0.025 m) of a 1M solution of sodium hexamethyldisilazide in THF. The solution was stirred for 20 minutes. A solution of 3.2 g (0.024 m) of pyrrolidine carbamoyl chloride in 10 ml of THF was added. The reaction mixture was allowed to warm to room temperature and was stirred for 48 hours. The solution was diluted with 100 ml of ethyl acetate and was washed with 1N HCl (3xc3x9775 ml), saturated NaHCO3 (1xc3x9775 ml), 1N NaOH (2xc3x9775 ml) and brine (1xc3x9775 ml) and was dried over MgSO4. The mixture was filtered and concentrated in vacuo and the residue was recrystalized from ethyl acetate/hexanes to give 8.6 g of a white solid.
300 MHz 1H NMR (CDCl3): xcex41.40 (s, 9H); 1.41 (s, 9H); 1.92 (m, 4H); 3.02 (d, 2H); 3.45 (t, 2H); 3.55 (t, 2H); 4.42 (dd, 1H); 4.99 (d, 1H); 7.05 (d, 2H); 7.15 (d, 2H).
Step C
N-FMOC)-(L)-4-(1-pyrrolidino-carbonyloxy)phenylalanine
The method of Reference Example 2 Step C was applied to 8.1 g (0.018 m) of the product of Step A to give 6.27 g of the title compound as a foam. 71% overall yield.
300 MHz 1H NMR (CDCl3): xcex41.97 (bs, 4H); 3.12 (bd, 2H); 3.4-3.6 (2 bm, 4H); 4.20 (m, 1H); 4.30-4.50 (m, 2H); 4.69 m, 1H); 5.59 (t, 1H); 7.00-7.42 (m, 8H); 7.55 (bm, 2H); 7.77 (d, 2H).
(L)-4-(2xe2x80x2-methoxyphenyl)phenylalanine, tert-butyl Ester Hydrochloride
To a solution of 4.85 g (0.0113 m) of the product of Reference Example 2 Step B in 100 ml of t-butyl acetate was added 5.53 g (0.056 m) of concentrated sulfuric acid. The solution was stirred at room temperature for 2 hours and then carefully neutralized by addition of saturated aqueous NaHCO3 solution. The solution was washed with NaHCO3 solution, dried over NaSO4, filtered and concentrated in vacuo. The residue was dissolved in 50 ml of ether and treated with anhydrous HCl gas with stirring to give a white precipitate. The solid was collected by filtration, washed with ether and dried in vacuo to give the desired product. 300 MHz
1H NMR (CD30D): 1.45 (s, 9H); 3.20 (d, 2H); 3.79 (s, 3H); 4.21 (t, 1H); 7.03 (m, 2H); 7.28 (m, 2H); 7.31 (d, 2H); 7.50 (d, 2H).
(L)-4-(2xe2x80x2,6xe2x80x2-Dimethoxyphenyl)phenylalanine, t-butyl Ester
To a solution of 18.5 g (55 mmol) of N-(Boc)-L-tyrosine, t-butyl ester (Reference Example 3, Step A) in 150 ml of dry methylene chloride was added 17.4 g (220 mmol) of pyridine followed at 0xc2x0 C. by the addition of 18.6 g (66 mmol) of triflic anhydride neat dropwise. The reaction mixture was stirred at 0xc2x0 C. and monitored by TLC. After 4 hours the mixture was diluted with 200 ml of methylene chloride and was washed with 1N HCl (3xc3x97100 ml), saturated sodium bicarbonate (2xc3x97100 ml) and brine (1xc3x9750 ml). The solution was dried over MgSO4, filtered and concentrated in vacuo to give an oil. The oil was dissolved in a mixture of 125 ml of toluene and 61 ml of ethanol. To this solution was added 11.3 g (62 mmol) of 2,6-dimethoxyboronic acid and 2.5 g of palladium tetrakistriphenylphosphine. The solution was treated with 18.3 g (133 mmol) of potassium carbonate dissolved in 30 ml of water. The mixture was heated to reflux over 4 hours, diluted with 200 ml of ethyl acetate and was washed with water (3xc3x9775 ml), brine (1xc3x9775 ml) and was dried over MgSO4. The mixture was filtered and concentrated in vacuo and the residue was purified by flash chromatography over silica gel eluted with a gradient of 5-20% EtOAc/hexanes to provide 14.7 g of N-(Boc)-(L)-4-(2xe2x80x2,6xe2x80x2-dimethoxyphenyl)phenylalanine, t-butyl ester as a white solid. The solid was dissolved in 350 ml of t-butyl acetate at 0xc2x0 C. and was treated with 8.3 ml of concentrated sulfuric acid. The cold bath was removed and after one hour TLC indicated only starting material was present. The reaction mixture was cooled in an ice bath once more and treated with 3.4 ml of concentrated sulfuric acid. The reaction was monitored by TLC. After consumption of the starting material the reaction mixture was diluted with 300 ml of ethyl acetate and was washed with 3xc3x97100 ml of 1N NaOH followed by brine (1xc3x97100 ml). The solution was dried over MgSO4. Filtered and was concentrated in vacuo to provide 8.9 g of (L)-4-(2xe2x80x2,6xe2x80x2-dimethoxyphenyl)phenylalanine, t-butyl ester. 500 MHz 1H NMR (CD3OD): xcex41.45 (s, 9H), 3.20 (d, 2H); 3.69 (s, 6H); 4.20 (t, 1H); 6.72 (d, 2H), 7.15 (m, 5H).
3(R)-amino-3-(4-biphenyl)propionic Acid, Methyl Ester
Step A
N-tert-Butoxycarbonyl-(S)-4-hydroxyphenylglycine
To a solution of (S)-(4-hydroxyphenyl)glycine (Sigma Chemical) (6.5 g, 39 mmol) in dioxane/water (1:1, 120 mL) was added triethylamine (5,9 g, 8.2 mL, 58 mmol) and [2-(tert-butoxycarbonyloxyimino)-2-phenylacetonitrile] (BOC-ON; 11 g, 45 mmol). After stirring overnight at room temperature, 300 mL of brine was added to the solution and the mixture was extracted with ether. (3xc3x97100 mL). The aqueous layer was acidified with HCl (pH=2) and extracted with 3xc3x97100 mL of ethyl acetate. The ethyl acetate layer was dried over MgSO4, filtered and the solvent removed under reduced pressure. The residue was chromatographed with 98/2 to 95/5 methylene chloride/methanol. Recovered 12 g of crude product. The impurity was removed following esterification of the product in the next step.
400 MHz 1H NMR (CDCl3): xcex41.37 (s, 9), 5.1 (1H, br s), 6.7 (d, 2H, J=8 Hz), 7.15 (d, 2H, J=8 Hz).
Step B
N-tert-Butoxycarbonyl-(S)-4-hydroxyphenylglycine, Methyl Ester
In a 50 mL round bottomed flask was added a 1:1 mixture of benzene:methanol and N-tert-butoxycarbonyl-(S)-4-hydroxyphenylglycine (2.8 g, 11 mmol). The solution was cooled to 0xc2x0 C. and a 2 M solution of trimethylsilyldiazomethane (Aldrich Chemical Co.) in hexane was added with vigorous stirring until a slight yellow color persisted. Then the reaction mixture solvents were removed under reduced pressure and the crude product was purified by flash chromatography (80/20 hexane/ethyl acetate) to give N-tert-butyloxycarbonyl-(S)-4-hydroxyphenylglycine, methyl ester (2.05 g, 7.3 mmol) (66% yield).
300 MHz 1H NMR (CDCl3): xcex41.43 (s, 9H), 3.71 (s, 3H), 5.22 (br d, 1H), 5.57 (1H, br d), 5.80 (br s, 1H), (6.7 (d, 2H, J=8 Hz), 7.17 (d, 2H, J=8 Hz).
step C
N-tert-Butoxycarbonyl-(S)-4-(trifluoromethylsulfonyloxy)phenylglycine, Methyl Ester
To a 25 mL round bottom flask fitted with a stir bar and septum was added N-tert-butyloxycarbonyl-(S)-4-hydroxyphenylglycine, methyl ester (1.9 g, 6.8 mmol) and pyridine (2.8 mL, 33 mmol) in 12 mL of methylene chloride. The flask was purged with N2, cooled to 0xc2x0 and trifluoromethanesulfonic anhydride (1.38 mL, 7.8 mmol) was added dropwise over several minutes, keeping the temperature at or below 4xc2x0 C. The solution was stirred for 1 h, then at room temperature for 4 h. The mixture was diluted with 20 mL of methylene chloride. The mixture was washed with 20 mL of 0.5 N NaOH, 1xc3x9720 mL of water and 2xc3x9720 mL of 10% citric acid. Dry the organic layer over MgSO4, filter, reduce the volume. Flash chromatography (75/25 hexane/methylene chloride) gave 2.3 g of desired product (81% yield).
300 MHz 1H NMR (CDCl3): xcex41.43 (s, 9H), 3.74 (s, 3H), 5.35 (1H, br d), 5.68 (br s, 1H), 7.27 (d, 2H, J=8 Hz), 7.47 (d, 2H, J=8 Hz).
Step D
N-tert-Butoxycarbonyl-(S)-(4-biphenyl)glycine
To a 25 mL round bottom flask fitted with a stir bar and septum was added N-tert-butyloxycarbonyl-(S)-4-trifluoromethylsulfonyloxyphenylglycine, methyl ester (690 mg, 1.67 mmol), anhydrous potassium carbonate (348 mg, 2.6 mmol) and benzeneboronic acid (411 mg, 3.4 mmol) in 15 ml of toluene and 3 mL of ethanol. The mixture was degassed under nitrogen with three freeze-thaw cycles and tetrakis(triphenylphosphine) palladium (94 mg, 0.085 mmol) was added to the reaction mixture and the mixture was heated between 75-90xc2x0 C. for 4 h. The solvent was removed under reduced pressure and the residue flash chromatographed with 85/15 hexane/ethyl acetate. Recovered 600 mg of the methyl ester (quantitative yield).
300 MHz 1H NMR (CDCl3): xcex41.44 (s, 9H), 3.75 (s, 3H), 5.37 (1H, br d), 5.62 (br s, 1H), 7.36 (m,.1H), 7.45 (m, 4H), 7.57 (m, 4H).
The ester was hydrolyzed with 1.2 eq of KOH in 10 mL of 4:1 ethanol:water (2 h). The solution was acidified with 2 N HCl (pH=2). Remove the solvents in vacuo and extract the free acid with methylene chloride. Recovered 430 mg of free acid (66% yield).
Step E
3-(N-tert-Butyloxycarbonyl)amino-1-diazo-3-(4-biphenyl)propan-2-one
To a 25 mL round bottom flask fitted with a stir bar and septum was added N-tert-butoxycarbonyl-(S)-4-biphenylglycine (430 mg, 1.31 mmol) in 10 mL of 2:1 methylene chloride:ether. The mixture was cooled to 0xc2x0 C. and N-methylmorpholine (159 xcexcl, 1.44 mmol) was added, followed by dropwise addition of isobutylchloroformate (179 xe2x96xa11, 1.38 mmol). The mixture was stirred for 1 h at 0xc2x0 C., then diazomethane in ether (excess, prepared from DiazaldR by literature procedure) was added dropwise to the reaction mixture. The mixture was stirred for 1 h then quenched with saturated sodium bicarbonate. The mixture was extracted with ethyl acetate. (2xc3x975 mL), washed with brine then dried over MgSO4. The mixture was filtered, the solvent removed under reduced pressure and the product isolated by flash chromatography (80/20 hexane/ethyl acetate) to give 280 mg (0.78 mmol) of product (58% yield).
300 MHz 1H NMR (CDCl3): xcex41.42 (s, 9H), 5.22 (bs, 1H), 5.29 (s, 1H), 5.9 (br s, 1H), 7.35-7.5 (m, 5H), 7.52-7.62 (m, 4H).
Step F
3(R)-amino-3-(4-biphenyl)propionic Acid, Methyl Ester
To a 25 mL round bottom flask fitted with a stir bar and septum was added (3-diazo-2-oxopropyl-1-(S)-(4-biphenyl))carbamic acid,tert-butyl ester (280 mg, 0.76 mmol), with 5 mL each of methanol and dioxane. The flask was cooled to 0xc2x0 C. and 0.15 eq (34 mg, 0.038 mmol) of silver benzoate in 500 xcexcl of triethylamine was added dropwise to the reaction mixture and the mixture allowed to stir at 25xc2x0 C. for 1 h. The reaction was worked up with 10% NH4OH in saturated NH4Cl (10 mL). Extract with ether (3xc3x9710 mL) and dry the organic layer over MgSO4. Filter, reduce the volume and flash chromatograph with 85/15 hexane/ethyl acetate. Recovered 260 mg of product (98% yield). Take this material and dissolve it in 10 mL of 1 N HCl in ethyl acetate. After stirring 2 h at room temperature, we obtained 180 mg of 3(R)-amino-(4-biphenyl)propionic acid, methyl ester hydrochloride. 300 MHz 1H NMR (CD30D): xcex42.90 (dd, 1H, J=18 Hz, J=6 Hz), 3.02 (dd, 1H, J=18 Hz, J=6 Hz), 3.66 (s, 3H), 5.9 (br s, 1H), 7.33-7.5 (m, 5H), 7.55-7.6 (m, 4H).
The following 3(R)-amino-propionic acid derivatives were prepared by the procedures described in Reference Example 6 substituting the appropriate boronic acid analog for benzeneboronic acid:
3(R)-amino-3-(4-(2xe2x80x2-methoxyphenyl)phenyl)propionic acid, methyl ester;
3(R)-amino-3-(4-(2xe2x80x2,6xe2x80x2-dimethoxyphenyl)phenyl)propionic acid, methyl ester.